Inositol polyphosphates (IPns) and phosphoinositides (PIPns) interact with over ten distinct families of binding proteins and three main classes of lipid-metabolizing enzymes. Success in the synthesis and use of affinity probes for isolating, labeling, and characterizing many PIPn and IPn interactive proteins now requires a new set of probes to understand the biological function of (P)lPn-protein interactions in regulating cell function. Therefore, in order to dissect the complexities of lipid signaling in living cells, we propose to prepare metabolically stabilized (ms) IPn and PIPn analogs as reporters of cell function. The goal is to develop both agonists and antagonists to regulate protein recruitment, and to prepare enzyme inhibitors and non-processable ligands to evade or block PIPn remodeling in living cells. In Aim 1, we will synthesize ms-(P)lPn analogs that fall into three classes: kinase-resistant, phosphatase-resistant, and phospholipase-resistant. These probes will be evaluated in Aim 2 as enzyme inhibitors and as (P)lPn-mimetics in biochemical and structural experiments. In Aim 3, we will determine the biological activity of ms-(P)lPn analogs as agonists or antagonists in molecular and cellular studies of changes in cell physiology. Initial validations will be conducted at University of Utah and through a consortium agreement with Utah State University. In addition, we will provide novel ms-(P)lPns to selected external collaborators, with whom we have jointly designed experiments and developed project aims. Finally, in Aim 4, we will prepare and characterize hybrid phosphatidylethanolamine-phosphoinositides capable of reporting on the importance of clustering and protein-lipid interactions in cells and model membrane systems. [unreadable] [unreadable]